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CRISPR-Casphi-based gene editing element and application thereof

A gene editing and component technology, applied in the fields of molecular biology and bioengineering, to achieve the effect of improving efficiency and improving delivery efficiency

Pending Publication Date: 2022-07-15
THE SECOND AFFILIATED HOSPITAL TO NANCHANG UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, affected by the transfection efficiency of AAV vectors into cells, the gene editing efficiency of delivering Cas9 gene fragments into cells with multiple AAV vectors is only between 9% and 59%. [9]

Method used

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  • CRISPR-Casphi-based gene editing element and application thereof
  • CRISPR-Casphi-based gene editing element and application thereof
  • CRISPR-Casphi-based gene editing element and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0182] Example 1: CRISPR-CasΦ-based gene cleavage single AAV delivery system

[0183] 1. Adeno-associated virus construction steps

[0184] (1) According to figure 1 As shown in D, construct an adeno-associated virus plasmid (crRNA sequences are shown in SEQ ID NO: 23 and SEQ ID NO: 24);

[0185] (2) using Escherichia coli strain DH5α to amplify the adeno-associated virus vector and the adeno-associated virus backbone vector plasmid;

[0186] (3) Use lipo3000 reagent to transfect the adeno-associated virus vector and the adeno-associated virus backbone vector plasmid into 293A cells, and replace the culture medium after 6 hours;

[0187] (4) Plaque test to observe the virus state, pick the plaque with the highest titer for follow-up experiments;

[0188] (5) The virus in the medium was added to the fresh 293A cell culture medium for a small amount of virus amplification. When the cells reappeared with plaques, the cells and supernatant were collected, and the virus was co...

Embodiment 2

[0198] Example 2: CRISPRa single AAV delivery system based on CRISPR-CasΦ

[0199] 1. Adeno-associated virus construction steps

[0200] (1) According to figure 2 As shown, construct adeno-associated virus plasmid (crRNA sequences are shown in SEQ ID NO: 25 and SEQ ID NO: 26);

[0201] (2) using Escherichia coli strain DH5α to amplify the adeno-associated virus vector and the adeno-associated virus backbone vector plasmid;

[0202] (3) Use lipo3000 reagent to transfect the adeno-associated virus vector and the adeno-associated virus backbone vector plasmid into 293A cells, and replace the culture medium after 6 hours;

[0203] (4) Plaque test to observe the virus state, pick the plaque with the highest titer for follow-up experiments;

[0204] (5) The virus in the medium was added to the fresh 293A cell culture medium for a small amount of virus amplification. When the cells reappeared with plaques, the cells and supernatant were collected, and the virus was collected by ...

Embodiment 3

[0214] Example 3: CRISPRi single AAV delivery system based on CRISPR-CasΦ

[0215] 1. Adeno-associated virus construction steps

[0216] (1) According to image 3 As shown in D, construct an adeno-associated virus plasmid (crRNA sequences are shown in SEQ ID NO: 27 and SEQ ID NO: 28);

[0217] (2) using Escherichia coli strain DH5α to amplify the adeno-associated virus vector and the adeno-associated virus backbone vector plasmid;

[0218] (3) Use lipo3000 reagent to transfect the adeno-associated virus vector and the adeno-associated virus backbone vector plasmid into 293A cells, and replace the culture medium after 6 hours;

[0219] (4) Plaque test to observe the virus state, pick the plaque with the highest titer for follow-up experiments;

[0220] (5) The virus in the medium was added to the fresh 293A cell culture medium for a small amount of virus amplification. When the cells reappeared with plaques, the cells and supernatant were collected, and the virus was collec...

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Abstract

The invention belongs to the technical field of molecular biology and bioengineering, and relates to a gene editing element based on CRISPR-Casphi and application of the gene editing element. Specifically, the invention relates to a gene editing element based on CRISPR-Casphi, a nucleic acid construct containing the gene editing element, a recombinant expression vector, a recombinant host cell and a gene editing method. According to the CRISPR-Casphi-based gene editing element disclosed by the invention, the crRNA and the target polypeptide can be jointly delivered by a single delivery system, so that the delivery efficiency of the crRNA and the target polypeptide for target gene editing is effectively improved, the gene editing efficiency is greatly improved, and the CRISPR-Casphi-based gene editing element has an important application prospect in the field of clinical gene therapy.

Description

technical field [0001] The present disclosure belongs to the technical field of molecular biology and bioengineering, and in particular, the present disclosure relates to a gene editing element based on CRISPR-CasΦ and its application. More specifically, the present disclosure relates to a CRISPR-CasΦ-based gene editing element, a nucleic acid construct comprising the gene editing element, a recombinant expression vector, a recombinant host cell, and a method for gene editing. Background technique [0002] The CRISPR / Cas system (Clustered regulatory interspaced short palindromicrepeat / Cas) is the adaptive immune system of archaea and bacteria. The CRISPR / Cas system can perform targeted editing of specific genomic loci, and has been widely used in gene editing experiments in many genera, including bacteria, fungi, Drosophila, nematodes, zebrafish, rats, mice, and humans. [0003] CRISPR sites and Cas genes are important components of the CRISPR / Cas system. CRISPR is a specia...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/55C12N15/62C12N15/113C12N15/864A61K48/00A61K38/46A61P35/00
CPCC12N9/22C07K14/4702C07K14/4705C07K14/4703C12N9/1007C12N9/78C12N15/113C12N15/86A61K48/0008A61K48/005A61K38/465A61P35/00C12Y305/04004C07K2319/09C07K2319/00C12N2310/20C12N2750/14143C12N2800/107
Inventor 黄凯李婧滢祝新跟罗敏涂泽伟雷锟坚龙小燕
Owner THE SECOND AFFILIATED HOSPITAL TO NANCHANG UNIV
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